Microfluidic droplet queuing network

ABSTRACT

A multi-port liquid bridge ( 1 ) adds aqueous phase droplets ( 10 ) in an enveloping oil phase carrier liquid ( 11 ) to a draft channel ( 4, 6 ). A chamber ( 3 ) links four ports, and it is permanently full of oil ( 11 ) when in use. Oil phase is fed in a draft flow from an inlet port ( 4 ) and exits through a draft exit port ( 6 ) and a compensating flow port ( 7 ). The oil carrier and the sample droplets ( 3 ) (“aqueous phase”) flow through the inlet port ( 5 ) with an equivalent fluid flow subtracted through the compensating port ( 7 ). The ports of the bridge ( 1 ) are formed by the ends of capillaries held in position in plastics housings. The phases are density matched to create an environment where gravitational forces are negligible. This results in droplets ( 10 ) adopting spherical forms when suspended from capillary tube tips. Furthermore, the equality of mass flow is equal to the equality of volume flow. The phase of the inlet flow (from the droplet inlet port ( 5 ) and the draft inlet port ( 4 ) is used to determine the outlet port ( 6 ) flow phase.

FIELD OF THE INVENTION

The invention relates to control of flow of small discrete quantities ofliquids (“droplets”) at the microfluidic scale.

PRIOR ART DISCUSSION

There are many applications emerging for the use of flowing liquiddroplets for uses such as chemical reactors. The liquid droplets may becontrolled in such a way that they are separated from one another by animmiscible carrier oil which also wets the inner channel or tubesurface. The droplets are thereby completely wrapped by the oil phaseand any chemical interaction with the surface, including carryover andcross-contamination between droplets is eliminated.

It is known to provide droplets by, for example, segmenting a singlephase homogeneous droplet into multiple smaller droplets of the samecomposition.

However the art provides little guidance for controlling flow ofmultiple droplets, particularly where droplets contain differentchemical compositions. WO20051002730 describes a microfluidic device inwhich droplets are provided by shearing force between an aqueous liquidfrom a channel and oil flowing in the channel into which the aqueousliquid enters.

The invention is directed towards providing improved control of suchdroplets.

SUMMARY OF THE INVENTION

According to the invention, there is provided a microfluidic network forqueuing a sequence of droplets in an immiscible carrier liquid, thenetwork comprising:

-   -   a draft conduit for flow of droplets with a carrier liquid;    -   at least one liquid bridge in the draft conduit, the bridge        having a chamber in which there is a draft inlet, a draft        outlet, an inlet port, and a compensation port;    -   wherein the inlet port is positioned for delivery of liquid to        be queued into the draft conduit so that said liquid flows from        the bridge in the carrier liquid in the draft conduit, and    -   wherein the compensation port is positioned for withdrawal of        carrier liquid to compensate for liquid added to the draft        conduit via the inlet port.

In one embodiment, the compensation port is configured to provide auniform target flow in the draft conduit.

In one embodiment, there are a plurality of bridges in the draft conduitand a liquid supply is connected to the inlet port of each bridge.

In one embodiment, the inlet port and the draft conduit are co-planar.

In one embodiment, the compensation port is at an angle to the plane ofthe inlet port and the draft conduit.

In one embodiment, the compensation port is at an angle of substantially90° to the plane of the draft conduit and the inlet port.

In one embodiment, the draft conduit inlet and an outlet to the bridgeare at approximately 120° to each other and the inlet port is in-planewith the draft conduit and at an angular separation of 120° from each ofthe draft inlet and outlet.

In one embodiment, there are a plurality of draft conduits in parallel

In one embodiment, the bridge inlet port diameter is in the range of 0.1mm to 0.6 mm.

In one embodiment, the bridge compensation port diameter is in the rangeof 0.1 mm to 0.6 mm.

In one embodiment, the separation of the compensation port from the axisof the draft conduit is in the range of 2 mm to 8 mm.

In one embodiment, the network further comprises a segmenter forsegmenting a large droplet or a stream into droplets and for deliveringsaid droplets to the inlet port of the bridge.

In one embodiment, the segmenter comprises a bridge having a chamberwith an inlet and an outlet, and said inlet and outlet are configured sothat a droplet temporarily adheres to the inlet and transfers to theoutlet when it becomes unstable.

In one embodiment, the segmenter comprises a chamber for containingcarrier liquid in the space between the inlet and the outlet.

In a further embodiment, the network comprises a plurality of bridges inthe draft conduit, at least one of said bridges being a mixing bridgedownstream of at least one other bridge, the mixing bridge comprisingmeans for mixing a droplet with a droplet flowing in the draft conduit.

In another embodiment, the mixing bridge comprises an inlet port for anadded droplet, configured for formation of droplets within its chamber,for contact and mixing of said droplets, and for transfer of the mixeddroplet to the draft outlet.

In one embodiment, the mixing bridge chamber is configured to fill withcarrier liquid to surround the droplets in the chamber.

In one embodiment, said supply comprises a well and a manifold fordelivering droplets from the well to a plurality of bridges.

In one embodiment, the network further comprises an infusion pump fordelivering carrier liquid to the manifold.

In one embodiment, the bridges and the draft conduit are arranged in anarray and there is a well adjacent each bridge.

In one embodiment, said wells are arranged in a pattern of an assay wellplate.

In a further embodiment, there are a plurality of wells and associatedmanifolds, and they are arranged for delivery of droplets of differenttypes to the bridges to achieve a serial flow of droplets of differenttypes in the draft conduit.

In one embodiment, the bridges are arranged so that droplets are addedsimultaneously at spaced-apart locations along the draft conduit.

In a further embodiment, the length of conduit between said supply andeach bridge is chosen according to modelling of an electric circuit, inwhich conduit length is equivalent to electrical resistance

In another aspect, the invention provides a method for managing a queueof droplets in any network as defined above, the method comprisingdelivering a sequence of droplets flowing in carrier liquid to eachbridge so that said droplets are added to carrier liquid in the draftconduit, carrier liquid is withdrawn via the compensation port of eachbridge to compensate for added liquid, and the separations of the inletdroplets are sufficient to achieve an adequate separation of droplets inthe draft conduit.

In one embodiment, there are a plurality of bridges in the draftconduit, and droplets of the same type are added substantiallysimultaneously to the draft conduit via the bridges so that there is asequence of droplets of similar type in the draft conduit.

In one embodiment, droplets of different types are added substantiallysimultaneously to the draft conduit, providing a sequence of droplets ofselected different types in the draft conduit.

DETAILED DESCRIPTION OF THE INVENTION Brief Description of the Drawings

The invention will be more clearly understood from the followingdescription of some embodiments thereof, given by way of example onlywith reference to the accompanying drawings in which:—

FIG. 1 is an illustration of a multi-port liquid bridge of a liquiddroplet network;

FIG. 2 shows queuing configuration of droplets in a tube in whichdroplets are grouped into sets of similar chemistry;

FIG. 3 shows queuing configuration of droplets in a tube in whichdroplets of different types are arranged in sequence;

FIG. 4 shows queuing configuration of droplets in a tube in whichdroplets are arranged in repeat sequence;

FIG. 5 shows time progression, left to right, of a queue formation ofdroplets with different chemistry in each;

FIG. 6 shows queuing of different droplets formed in identical parallellines;

FIG. 7 shows a queuing system having an upstream liquid bridge segmenterto form droplets, queuing bridges, and a downstream bridge mixer to addchemistry to each of the queued droplets;

FIG. 8 shows network having wells, manifolds, segmenter bridges, andqueuing bridges to produce a queue of droplets with different chemistryin each;

FIG. 9 shows the physical construction of the bridge of FIG. 1;

FIG. 10 shows a bridge having three in-plane with three ports at 120° toeach other and a compensating port at 90° to this plane;

FIG. 11 is an external perspective view of an array plate of a network;

FIG. 12 shows junction caps and a fluorinated ethylene propylene (FEP)well inserts of the well plate, and

FIG. 13 is a more detailed perspective view;

FIG. 14 is a perspective view of a full microfluidic system;

FIG. 15 shows a syringe pump system which supplies the system with fluidflow;

FIGS. 16 and 17 shows a multiple syringe and manifold infuse array inuse;

FIG. 18 shows a manifold used to convert fluid flow into multiple equallines of fluid flow; and

FIG. 19 is a diagram illustrating operation of electrical analysissoftware modelling microfluidic networks.

DESCRIPTION OF THE EMBODIMENTS

If liquid droplets are added to a tube or channel at different axiallocations then the flow rate and velocity would increase in the flowdirection, giving an undesirable summing of flow rates when queuing anumber of droplets. To overcome this, equal volumes of liquid are addedand subtracted simultaneously to keep the axial velocity in the queuingtube constant. A droplet network has bridges such that wherever adroplet is added to a draft flow excess carrier liquid (silicone oil) isremoved via a compensating port. An aqueous phase entering from an inletport will be delivered to an exit port, likewise for an aqueous phasearriving at an upper inlet port. By this means aqueous droplets can beintroduced into the draft stream and delivered straight through all ofthe downstream bridges. In a single device, with steady inlet flows, aqueue of droplets can be formed and delivered. Furthermore, a downstreamsegmenter can then be used to break a stream of droplets into dropletsof a different size.

A multi-port liquid bridge 1 is illustrated in FIG. 1. Arrows indicateflow direction. Aqueous phase droplets 10 are distinctly shown with anenveloping oil phase carrier liquid 11, implied in all of the drawings.A chamber 3 links four ports, and it is permanently full of oil 11 whenin use. Oil phase is fed in a draft flow from an inlet port 4 and exitsthrough a draft exit port 6 and a compensating flow port 7. The oilcarrier and the sample droplets (“aqueous phase”) flow through the inletport 5 with an equivalent fluid flow subtracted through the compensatingport 7. The latter is perpendicular to the plane of the page, and themanner of representing this used in FIG. 1 is also used in the remainderof the drawings.

The ports of the bridge 1 are formed by the ends of capillaries held inposition in plastics housings, as described in more detail below. In oneembodiment the inner diameter of the chamber is 2.4 mm and the height ofthe chamber is 2.5 mm. The ports 4-7 are 0.4 mm in diameter andgenerally in the order of 0.1 to 0.6 mm in diameter.

The phases are density matched. Density matching creates a nearweightless environment where gravitational forces are negligible. Thisresults in droplets 10 adopting spherical forms when suspended fromcapillary tube tips. Furthermore, the equality of mass flow is equal tothe equality of volume flow. The phase of the inlet flow (from thedroplet inlet port 5 and the draft inlet port 4) is used to determinethe outlet port 6 flow phase.

FIG. 2, FIG. 3 and FIG. 4 represent a range of queuing configurations ofdroplets in a tube. FIG. 2 shows droplets grouped into sets, FIG. 3shows droplets arranged in sequence, and FIG. 4 shows droplets arrangedin repeat sequence.

A queuing network is illustrated in FIG. 5, which shows a column ofthree bridges 1 at four instances in time, left to right. At a time t₁the droplets taken from a well are approaching the bridges from the leftinlet port 5 in phase. At time t₂ the droplets bridge to the lower port6. The compensating port 7 is out of plane and too far from any inlet tobridge with the droplets, it therefore only takes the oil phase. At timet₃ the droplets are shown queued in the draft tube. At time t₄ the inletport 4 is shown bridging with the outlet port 6, and therefore anydroplet that enters from the top, or from the left, always exits fromthe exit port 6. A queue of different droplets of the aqueous phase isthereby formed. Operation of the compensating port 7 ensures uniformityof flow down through the draft conduits 4, 6.

Many lines can be configured in parallel as shown in FIG. 6 to give ahigh-throughput system. This is particularly useful for managing queuingof droplets as micro-reactors for thermal cycling through multiplethermal cyclers in parallel. This is very advantageous for achieving ahigh throughput, and it avoids need to cyclically heat and cool reactionvessels.

A network which performs segmentation of a continuous aqueous phase intodroplets, and also queuing and mixing of the droplets, is shown in FIG.7. This embodiment shows how the queue bridge configuration of FIG. 5can be integrated with other bridges, and how this network can bearranged in parallel. In the arrangement of FIG. 7 there aresegmentation bridges 20 having ports 21 to 24, but only ports 21, 23 and24 are operational as there is no flow through port 22. The bridges 20are arranged upstream of the queuing system, one for each well, tosegment the continuous phase drawn from a well. The segmentation isachieved by a droplet forming within a chamber between the ports 23 and24, and due to inter-facial tension and immiscibility between theaqueous and oil phases, breaking off and entering the port 24.

A single bridge 1 operating as a mixing bridge is arranged downstream ofeach draft flow, so that a single phase from a well X is segmented andthen mixed with each of the queued droplets A B, . . . in succession inthe mixer bridge 1. The mixer bridges 1 are arranged so that there issimultaneous arrival of droplets at them. This will also work if thephase from well X is continuous. Within each mixer bridge, droplets format the ends of the ports 4 and 6, they mix due to their internalpressures and inter-facial tension, and the mixed droplet exits via theexit ports 6.

The end result is the delivery of a queue of mixed droplets which flowdownstream to be further processed by, for example, a thermal cycler. Anapplication of this is the arrangement of primers in a well for queuing,then the addition of a patient's sample along with the other requiredpremix occurring in the mixer. A continuous thermal cycler for DNAamplification using the polymerase chain reaction (PCR) may bedownstream from the mixers.

Referring to FIG. 8, a network has two wells 30 and manifolds 31 toproduce a queue of droplets with alternative chemistry in each. Themanifolds 31 distribute the flow to segmenter bridges 20 so that eachline carries the same flow rate. Downstream of the segmenter bridges 20are the queue-bridges 1 which operate in the same way as those shown inFIG. 5.

FIG. 9 is a three dimensional view of the bridge 1. The bridge chamber3, the upper draft inlet 4, left droplet inlet port 5, lower draft exit6 and the compensation port 7 are all clearly illustrated. The outercylinder is a solid plastics wall, in this case polycarbonate.

However, other configurations are possible, and FIG. 10 illustrates oneexample in which three in-plane ports 41, 42 and 43 are at an equalangular separation of 120°, and the compensating port 44 is again at90°.

In one embodiment the tubing used for the fluid flow has an exteriordiameter of 0.8 mm and an interior diameter of 0.4 mm, the distancebetween bridges is 9 mm, the bridge chamber is of diameter 2.4 mm, andthe outer diameter is approximately 6 mm.

In one embodiment, the flow rates and droplet volumes for the apparatusas follows, with reference to the bridge 1: flow rate in the draft inlet4, the droplet inlet 5, the draft exit 6, and the compensating exit 7are all equal (i.e. q₄=q₅=q₆=q₇); the sum of the inlet flow rates isalso equal to the sum of the outlet flow rates (q₄+q₅=q₆+q₇); droplet orplug volumes are between 30-300 nl; and volumetric flow rate is 3 μl/minwith velocities of the order 1 mm/s. These are the conditions used whenthe queue is used to array primers upstream of a PCR DNA amplifier.

A network may segregate primers into a controlled and orderly flow ofdroplets. Multiple wells of differing primers feed into a single tube ofmain fluid flow. With an applied force to the primers in each well, aset of primer droplets will be formed at each bridge in the tube of mainfluid flow. This can be used to give a very consistent and predictableflow of queued primer droplets. It will be appreciated that multiplewells in multiple parallel tubes of fluid flow can achieve a largenumber and/or arrangement of differing primers. A number of well arrayplates can be used in parallel to acquire a sufficient number of primersfor a required DNA test. Groups of multiple droplets (approximately 10)are used to increase the sample size and thus increase the certainty ofthe final test results. The queuing system receives fluid flow from theinfuse manifold of the pumping system, flows through a distribution headinto a queuing cartridge and fluid is then withdrawn from the queuingcartridge, through the distribution head and back to the withdrawalmanifold of the pumping system.

As illustrated in FIGS. 11 and 12 a plate 50 has integrally moundedbases 51 for wells and 52 for bridges. A cap 53 is provided to completea bridge 63 (shown in FIG. 13) and a well insert 54 is provided tocomplete each well 64 (also shown in FIG. 13). FIG. 13 shows capillaries61 interconnecting the wells 64 and the bridges 63, all in a queuingnetwork cartridge 60. The draft flow is along each line of bridges 63.The overall configuration mimics that of a conventional 96-well assayplate, and so samples can be dispensed by conventional dispensingequipment onto the wells 51. The cartridge 60 consists of fluoropolymertubing 61 and three injection moulded parts: a polycarbonate array plate62, 48 polycarbonate caps 53 and 48 fluorinated ethylene propylene (FEP)wells 54. All parts are manufactured using standard injection moulding.Gate and ejector pin locations were placed at sites that did notinterfere with the operation of the components. A flatness tolerance of200 micrometers was applied to the upper face of the array plate toensure a uniform interface with mating fluidic ports.

A complete microfluidic system is depicted in FIG. 14. A distributionhead 71 containing mating ports is milled from polycarbonate. A verytight flatness tolerance is applied to the lower surface of thedistribution head 71 in order to mate with the upper surface of thearray plate 60. A stepper motor 72 with gearing 73 which is situated ona gear train mount 74 is used to drive a cradle base 75 containing thequeuing cartridge 60 against a support head 76 containing thedistribution head 71. By doing so the necessary connections are madebetween the queuing cartridge 60 and the distribution head 71. The geartrain mount 74, the cradle base 75 and the support head 76 are all madefrom aluminium. Steel bars 77 and collars 78 are used to position theplates and act as guides. Bearings 79 on the cradle base 75 allow formotion to and from the distribution head 71. The queuing cartridge sitson a cradle 80 which is fitted onto the cradle base. The cradle 80 is atwo-part piece with the top half manufactured from polycarbonate and thebottom half manufactured from aluminium. The rotational force of themotor 72 is converted to an upwards force acting on the array platethrough a gear and cam mechanism 73

Assembling the queuing cartridge 60 involves fastening the polycarbonatecaps 53 and the FEP wells 54 to the appropriate locations. Contactbetween mating parts is made via a compression press-fit between thepins on the array plate 60 and the holes on the well and caps. Shortlengths of rigid PEEK tubing are then inserted into the withdrawal portsof the array plate. These lengths of tubing provide a cylindricalgeometry for spacing the tubing tips. The microfluidic network of tubing61 is then formed by placing tubing in the appropriate ports. The tubing61 connecting each bridge typically measures 8 mm. The tubing 61connecting the wells to the bridges typically measures 12 mm. Sealing ofthe tubing network is achieved with the use of a poly dimethyl siloxane(PDMS) encapsulant. This encapsulant is mixed as a two-part resin,degassed in a vacuum chamber and poured into the array plate cavity. Theassembly is then cured in an oven at 80° C. for 1 hour. The curedencapsulant forms an elastomeric seal to ensure primer and oil flow onlythrough tubing and not between array plate-tubing interfaces. Finally,the PEEK tubing used to space the tubing tips is removed.

Assembly of the distribution head involves the connection of tubing tothe appropriate connectors. Again sealing of the tubing network isachieved with the use of a poly dimethyl siloxane (PDMS) encapsulant.The same method of mixing, pouring and curing of the PDMS as mentionedabove is used.

The distribution head performs the task of distributing the flow fromthe 48 tubes of the manifold system to the top of the wells on thequeuing cartridge. This fluid flow from the manifold system, through thedistribution head to the top of the well, is used to pump the primerfrom the wells down the connecting tubes and into the bridges.

The assembled queuing cartridge 60 and distribution head 71 are primedwith AS100 silicone oil prior to first use. This step removes trappedair from tubing and liquid bridge cavities. Primer is then loaded intowells of the queuing cartridge via a standard pipette. The pipette tipis submerged under the level of the oil and in contact with the throatof the well such that the sample is transferred directly into thetubing. Any backflow thereafter is accommodated by the expanding conicalsection of the wells. Care must be exercised not to introduce largequantities of air into the tubing after the primer has been infused. Thecartridge is then ready to be loaded into the cradle with thedistribution head to be loaded into the support head.

The queuing cartridges 60 are designed to supply enough primer for anumber of tests. After the cartridge is depleted of primers, either anew cartridge can be used or the old cartridge can be refilled. Thedistribution head, platform plates and motor system are all permanentfeatures of the system.

There are many ways of pumping fluid through the system. A singularplate with indentations can be used to feed oil flow into the queuingcartridge. By placing the plate in a bath of oil into the queuingsystem, the compression force applied to the queuing system can create aconstant fluid flow. Also a multiple syringe system can be applied inorder to get a multiple fluid flow through tubing into the queuingsystem. However the current design uses syringe pumps to deliver thenecessary flow to the input lines of the queuing system. It is doneeither directly or by back-pressuring a storage well. For the multipleline fluid flow a limited number of syringes are used to pressurize areservoir or manifold with many outlets. A Harvard pump withstepper-motor drive is used to drive the limited number of syringes.

Motor systems as shown in FIG. 15 give a constant feed rate to infuseand withdraw oil phase from the queuing system 70. The motor is astepper motor 100 driving a lead screw 101 via a belt 102. The rotatinglead screw drives a pusher block 103 which in turn applies a constantvelocity to the syringes 104. The constant flow from the infuse syringesis pumped through manifolds 105 as depicted in FIG. 16 which willseparate the fluid flow into multiple tubes with equal flow rates. Thefluid flow then enters the queuing system. The fluid is then withdrawnat the same flow rate using a similar motor system in reverse. Amanifold 105 is now used to reduce the number of withdrawal tubes whichis in turn connected to withdrawal syringes as shown in FIG. 17. Thissystem uses multiple syringes for infuse and withdrawal, however asingular syringe with a larger manifold can also achieve the same task.

The manifold 105 is shown in more detail in FIG. 18. It is milled frompolycarbonate. The tubing is cut square. Equal length tubing is insertedinto holes in the manifold and are positioned flush with an insidechamber 106. A spare tube is inserted flush with the surface at aninverted cone section 107 of the chamber. This inverted cone collectsthe trapped air in the system and allows for the air to be drained viathe spare tube. The tubes are then held in place with PDMS. Again thesame mixing, pouring and curing method as in the queuing system areused. A cap array 108 with PEEK tubing flush with the inside surface ispress fitted into the opposite end of the chamber. The cap is then fixedin place with epoxy encapsulant. This encapsulant is mixed as a two-partresin, degassed in a vacuum chamber and poured into the array platecavity. The assembly is then cured in an oven at 50° C. for 1 hour. Thecured encapsulant forms a solid seal to ensure no leaks occur to thechamber.

Syringes 109 are placed on top of the motor system and the manifold PEEKtubing is connected. The entire tubing array of the pumping system isprimed prior to the tubes exiting from the manifold are connected to thequeuing system. Priming the system involves driving fluid through thesystem and then draining the air from the system via the spare tubing ineach chamber. This must be done before the pumping system is ever used.After the initial priming, the system should not need to be primed againunless air is trapped in the tubing. The same process must be thenrepeated. After the fluid has run through the system the flow is thenwithdrawn from the system back through the withdrawal manifolds, intothe withdrawal syringes which are attached to another motor system forwithdrawal. At present the infuse and withdrawal are powered by separatemotors however since the infuse and withdrawal are at the same flow ratethe same can be achieved from a single motor adapted to suite bothinfuse and withdrawal.

In summary, the pumping mechanism of FIGS. 16 and 17 pump oil throughthe manifolds 105, and from there into the system 70 of FIG. 14. Withinthis system the liquids are processed in a network such as illustratedin FIGS. 5 to 8.

Referring to FIG. 19, a queuing system employing a pressurized source,or sources, to address multiple pressurized lines or wells may bemodeled by drawing an electrical analogy with fluidic and geometriccharacteristics of the system.

The following may be regarded as equivalent electrical and fluidicparameters:

-   -   Electrical Resistance, R=Fluidic Resistance,

$( \frac{8\;\mu\; L}{\pi\; R^{4}} ),$where μ denotes fluid viscosity, L the conduit length and R the conduitradius.

-   -   Electrical Current, I=Fluid Flowrate, Q.    -   Voltage Drop, ΔV=Pressure Drop, ΔP.

The electrical analogy permits the use of electrical engineeringsoftware to model a droplet network. Hence, electrical engineeringsoftware may be used to predict theoretically correct flowrates in everypressurized line within a microfluidic network. FIG. 19 presents asection of an electrical circuit used to model a microfluidic network.The correct selection of electrical resistance within each branch of thecircuit may be used to define appropriate lengths and radii of conduits.For example, the tubing leading from the manifold to the upstreambridges needs to be longer than that leading to downstream bridges, inproportion to the lower resistances illustrated.

It will be appreciated that a network of the invention allows for aqueue of aqueous droplets to be formed with a different chemicalcomposition in each droplet. Serial line of bridges can be arranged inparallel to give a high throughput. A network may have a segmenter sothat plugs of aqueous phase are formed upstream of the queuing devicesAlso, liquid bridge mixers may be provided downstream of each serialline for adding chemical or biological samples to the queued droplets. Anetwork may be fed from a small number of wells through a manifold togive a queue of droplets which differs from the one given above, forexample, with every other droplet having a different chemistry. Thenetwork may be manufactured to have a simple geometry of bridges thatcan be connected together in a variety of ways with interconnectingcircular tubing.

The invention is not limited to the embodiments described but may bevaried in construction and detail. It will be appreciated by personsskilled in the art that variations and/or modifications may be made tothe invention without departing from the scope of the invention.

The invention claimed is:
 1. A method for managing a queue of dropletsin a network, the method comprising: delivering a sequence of dropletsflowing in a carrier liquid to at least one junction, wherein the atleast one junction comprises: at least one sample inlet, wherein thesequence of droplets and carrier liquid enter the junction; at least onecarrier liquid inlet wherein additional carrier liquid enters thejunction; a first carrier liquid outlet wherein the sample and carrierliquid exit the junction; a second carrier liquid outlet wherein onlycarrier liquid exits the junction; withdrawing carrier liquid from thejunction through the second carrier liquid outlet; withdrawing thesequence of droplets and carrier liquid from the junction through thefirst carrier liquid outlet, wherein the exiting sequence of dropletsare sufficiently separated.
 2. The method of claim 1 wherein thejunction is a liquid bridge.
 3. The method of claim 1 wherein the methodfurther comprises delivering a sequence of droplet through more than onejunction.
 4. The method of claim 3, wherein the more than one junctionsare in series.
 5. The method of claim 3, wherein the more than onejunctions are in parallel.
 6. The method of claim 1 wherein the carrierliquid is an oil.
 7. The method of claim 1 wherein the second carrieroutlet is a compensation outlet.
 8. The method of claim 1 whereincarrier liquid enters the junction from the carrier liquid inlet andexits the carrier liquid outlet at the same rate.
 9. The method of claim1 wherein the method includes processing the sequence of droplets. 10.The method of claim 9 wherein the processing includes thermal cyclingthe droplet.
 11. The method of claim 9 wherein the processing includesanalyzing a droplet of the sequence of droplets for the presence orabsence of a target nucleic acid.
 12. A method of forming dropletshaving varying chemistries comprising: delivering a first sequence ofdroplets flowing in a carrier liquid to a first junction wherein thefirst junction comprises: at least one sample inlet, wherein thesequence of droplets and carrier liquid enter the junction; at least onecarrier liquid inlet wherein additional carrier liquid enters thejunction; a first carrier liquid outlet wherein the sample and carrierfluid exit the junction; a second carrier liquid outlet wherein onlycarrier fluid exits the junction; delivering a second sequence ofdroplets to a second junction wherein the second junction comprises: atleast one sample inlet, wherein the sequence of droplets and carrierliquid enter the junction; at least one carrier liquid inlet whereinadditional carrier liquid enters the junction; a first carrier liquidoutlet wherein the sample and carrier fluid exit the junction; a secondcarrier liquid outlet wherein only carrier fluid exits the junction;adding a first primer to the first sequence of droplets at the firstjunction to form a first mixed droplet; adding a second primer to thesecond sequence of droplets at the second junction to form a secondmixed droplet; withdrawing carrier liquid from the first junction andthe second junction through the second carrier liquid outlet;withdrawing the sequence of droplets and carrier liquid from the firstjunction and the second junction through the first carrier liquid outletflowing the first mixed droplet along a first path and flowing thesecond mixed droplet along a second path, wherein the first primer andthe second primer are different primers, and wherein the carrier liquidentering the first junction and the second junction is withdrawn at thesame rate.
 13. The method of claim 12 wherein the first path and thesecond path are the same path.
 14. The method of claim 12 wherein thefirst junction is a liquid bridge.
 15. The method of claim 12 whereinthe second junction is a liquid bridge.